Rabbit anti pr

To investigate NPY^ARN co-expression with gonadal steroid hormone receptors, free-floating immunohistochemistry was performed on coronal sections throughout the rostral, middle, and caudal ARN (rARN, mARN, cARN). NPY^ARN neuron somata were labeled for GFP reporter expression using a chicken anti-GFP primary antibody (1:5000; Aves Labs) [45 (link)] as above. Every third section was co-labeled for one of the following steroid hormone receptors: progesterone receptor (rabbit anti-PR, 1:100; Dako (Agilent), CA, USA; VEH n = 8, PNA n = 10; RRID:AB_2315192) [46 (link)], estrogen receptor α (rabbit anti-ERα, 1:5000; Millipore; VEH n = 5, PNA n = 5, RRID:AB_310305) [47 (link)], or androgen receptor (rabbit anti-AR PG-21, 1:200; Millipore, MA, USA; VEH n = 4, PNA n = 4; RRID:AB_310214) [48 (link)]. To amplify GFP signal, a goat anti-chicken AlexaFluor488 antibody was used (1:200; Molecular Probes, OR, USA), while steroid hormone receptors were labeled using a goat anti-rabbit AlexaFluor568 antibody (1:200; Molecular Probes, OR, USA).

For histological examination, X-gal + glands were embedded in paraffin and cut in 5 μm sections and mounted on positively charged slides. Sections were subsequently cleared in xylenes and rehydrated through ethanol gradients. Antigen retrieval was performed by heating slides in a boiling water bath for 20 min. in either 10mM citrate buffer pH 6.0 (Dako, Capenteria, CA) or Tris-EDTA pH 9.0 (Dako). Endogenous peroxidase activity was blocked by using 3% hydrogen peroxide for 15 minutes at room temperature. Slides were blocked with normal horse serum (Vector Laboratories, Burlingame, CA) for 1 hour at room temperature and then incubated overnight with primary antibodies at 4°C. Primary antibodies used were rabbit anti-PR (1:150; Dako), rabbit anti-ERα sc#-542 (1:75; Santa Cruz Biotechnology, Dallas, TX). Secondary antibody staining was performed using the R.T.U. Vectastain (goat anti-rabbit/mouse) kit (Vector Laboratories). Staining was visualized using the DAB peroxidase substrate kit (Vector Laboratories) per manufacturer's recommendations. Slides were counterstained with Mayer's hematoxylin (Sigma-Aldrich) and negative tissue controls were included in all immunohistochemical analyses.